www.euacademic.org
Brine Shrimp Bioassay of Plants of the Brazilian Amazon
Rainforest
ANDRÉ CAMARGO DE OLIVEIRA
Instituto Nacional de Pesquisas da Amazônia - INPA, Amazonas, Brazil
Universidade Estadual de Roraima - UERR, Roraima, Brazil DENNY WILLIAM DE OLIVEIRA MESQUITA Instituto Nacional de Pesquisas da Amazônia - INPA, Amazonas, Brazil Universidade Federal de Rondônia - UNIR, Campus Cacoal, Rondônia, Brazil
Universidade Federal do Amazonas - UFAM, Amazonas, Brazil ADRIANA SPIROTTO STEIN MESQUITA1 Instituto Nacional de Pesquisas da Amazônia - INPA, Amazonas, Brazil
Universidade Federal do Amazonas - UFAM, Amazonas, Brazil CARROMBERTH CARIOCA FERNANDES Instituto Nacional de Pesquisas da Amazônia - INPA, Amazonas, Brazil Universidade Federal do Acre - UFAC, Acre, Brazil
CARLOS CLEOMIR DE SOUZA PINHEIRO Instituto Nacional de Pesquisas da Amazônia - INPA, Amazonas, Brazil EVELYSE SOARES DE SOUZA Instituto Nacional de Pesquisas da Amazônia - INPA, Amazonas, Brazil Universidade Federal do Amazonas - UFAM, Amazonas, Brazil
MARIA IZABEL CORREIA OSORIO Instituto Nacional de Pesquisas da Amazônia - INPA, Amazonas, Brazil LORENA MAYARA DE CARVALHO CURSINO Instituto Nacional de Pesquisas da Amazônia - INPA, Amazonas, Brazil Universidade Federal do Amazonas - UFAM, Amazonas, Brazil JANE VASCONCELOS NEVES MARINHO Instituto Nacional de Pesquisas da Amazônia - INPA, Amazonas, Brazil Universidade Federal do Amazonas - UFAM, Amazonas, Brazil MERTILHA MURARI BRENNER BELESA Instituto Nacional de Pesquisas da Amazônia - INPA, Amazonas, Brazil IEDA LEÃO DO AMARAL Instituto Nacional de Pesquisas da Amazônia - INPA, Amazonas, Brazil
CECÍLIA VERÔNICA NUNEZ Instituto Nacional de Pesquisas da Amazônia - INPA, Amazonas, Brazil
Abstract
This article describes the evaluation of the toxic potential of extracts of plant species Amazon using the brine shrimp lethality bioassay method. Many studies in the literature on phytochecial do not describe the biological activities of any kind. The test for the assessment of the toxicity was performed using larvae of Artemia salina with 48 h of hatching, the type nauplii, in concentrations of 1000, 100 and 10 µg.mL-1. Was observed that, of the species that have been assessed, the vast majority has the potential cytotoxic activity, being that few extracts showed inactivity. Currently searching for substances with pharmacological activities promising is the first step in the development of new drugs from plants.
Keywords: cytotoxic activity, Artemia salina, amazonian species, Capparaceae, Cecropiaceae, Fabaceae, Olacaceae, Rutaceae, Salicaceae, Verbenaceae.
1. Introduction
The plants are the main sources of natural products biologically active. A plant produces thousands of different substances, being only a small part responsible for their pharmacological activity. Many natural products are characterized and published without its biological activity has been tested. Many times this is due to the cost or complexity of the test.
The search for substances with potential biological activities, in their vast majority, it is guided by information in respect of the use of certain plants in folk medicine, chemotaxonomic information or even for biomonitoring, but with the small number of plants studied scientifically, it is very likely that new substances with promising potential to be discovered so completely random, due to research in plants little known or difficult to access.
In recent years, has been a great interest in alternative therapies and therapeutic use of natural products, especially those that are derived from plants. This interest in drugs originating in plants has several reasons, you can quote among the main ones: a) conventional medicine can often be inefficient (low effects on cure or therapy ineffective); b) high side effects of conventional drugs; (c) misuse and/or incorrect synthetic drugs, resulting in resistance to drugs (Peet, 2010); (d) a large percentage of the world's population does not have access to conventional pharmacological treatment; e) the popular medicine suggests that natural products are dressings (Zomlefer, 1994).
The test of lethality against the microcrustacean Artemia salina is a simple test at low
cost, and the results from this test are correlated with the cytotoxicity of some types of cancer cells (Grayer & Kokubun, 2001), with insecticides (Kraft et al., 2000) in addition to cytotoxic activities (Meyer et al. , 1982).
In this work we investigated the cytotoxic potential of some Amazonian species, using
the brine shrimp lethality bioassay method with Artemia salina Leach. The bioassay
was chosen because it is easy to perform and the microcrustacean of easy reproduction. Were employed different dosages of extracts, quantifying the number of individuals live and dead individuals (Raven et al., 1996).
2. Material and Methods
The test for the assessment of the toxicity was performed according to the methodology described by McLaughlin and collaborators (1995). The plant material was dried, pulverized and through extraction from cold with solvents of different polarities (dichloromethane, methanol and water) were obtained the plant extracts that were subjected to the test of lethality front to Artemia salina.
Eggs of Artemia salina were placed to erupt in a solution of sea salt (38 g.L-1), a small
The obtained extracts of plant materials studied were weighed (20 mg) and diluted in 2,0 mL of methanol, DMSO or water, depending on the type of extract. From this stock solution (10
mg.mL-1), two other dilutions were carried out in order to obtain solutions with concentrations
of 1 and 0,1 mg.mL-1. Each extract was tested in triplicate, and each well was added 500 mL of
the stock solution of extract, 10 larvae of Artemia salina and the solution of sea salt to adjust
the volume to 5 mL, resulting in solutions of concentrations of 1000, 100 and 10 kg.mL-1. The
nine bottles tests and a bottle of white control were rested and discovered and, after 24 hours was the count of the number of surviving larvae (Figure 1). The data obtained were statistically analyzed and was calculated based on the LC50 of the extracts. LC50 is the
concentration lethal to 50% of the organisms. Positive result of this test indicates potential toxic activity of bioactive compounds in plant extracts.
Artificial lighting with fluorescent lamp; Ambient temperature; Aeration: air pump. Becker 500 mL.
Figure 1. Scheme of the brine shrimp lethality bioassay method (from Mesquita et al., 2015).
Figure 2. Assay being performed.
Eggs of Artemia salina
Aqueous sea salt 38 g.L-1
Hatching eggs
48 h
10 Larvae per well CONTROL
(Larvae + solvent) EXTRACT*
(1000 g.mL-1)
EXTRACT* (1000 g.mL-1) CONTROL
(Larvae + saline solution)
Count of surviving larvae
24 h
EXTRACT* (1000 g.mL-1)
* Diluted in DMSO
3. Results and Discussion
The bioassay front to the larva of microcrustacean Artemia salina has been used as an
indicator of toxicity for both screening for substances with activity anti-tumor, as for
substances with pesticide activity and cytotoxic (McLaughlin et al., 1995). This test is
considered to be a quick test, cheap and very easy to be implemented. Also called "Brine Shrimp Letality Test" (BST), the test has the advantage of using individuals living well small and in small quantity. LC50 values < 1000 g.mL-1 are considered significant for crude extracts
(Parra et al., 2001). This bioassay shows a positive correlation with tests on cells
9 kb (nasopharyngeal carcinoma human p = 0.036 and kappa = 0.56), where the values of
ED50 for cytotoxicity are generally close to a tenth part of LC50 values found in
bioassays with Artemia salina, as observed by McLaughlin and
collaborators (1995) and cytotoxicity on cancerous cells P-388, observed by Meyer and collaborators (1982).
Table 1 illustrates the species used in the test as well as the CL50 obtained and their
respective activities. Was observed that, of the species that have been assessed, the vast majority has the potential cytotoxic activity, being that few extracts showed inactivity. The results obtained have been a sign of departure for future phytochemical studies and other biological activities.
Table 1. Results of the species tested front of lethality against Artemia salina. PLANT FAMILY
Scientific Name Part used
CL50 (g.mL-1) DCM MeOH H2O
CAPPARACEAE
Crateva benthamii Peel 410 520 660
CECROPIACEAE
Cecropia purpurascens, Leaves 230 280 490
FABACEAE
Campsiandra laurifolia
Leaves 290 330 560
Branches 450 760 990 ( -) FABACEAE
Deguelia duckeana Roots 230 590 870
OLACACEAE
Minquartia guianenis Leaves 250 320 790
RUTACEAE
Zanthoxylum sp Leaves 250 480 720 SALICACEAE
Salix martiana Seeds 670 790
980 ( -) VERBENACEAE
Vitex cymosa Roots 250 420 610
* Legend: ( - ) = considered inactive; DCM = dichloromethane extract; MeOH = methanol extract ; H2O = aqueous extract.
4. Conclusion
important to emphasize that the lethal test front to Artemia salina, in addition to simple and easy to be implemented is pretty accurate.
Acknowledgements
A. C. Oliveira thanks FAPEAM by stock exchange of DCR and financial support. D. W. O. Mesquita is grateful to CNPq for masters scholarship and CAPES of doctorate scholarship and A. S. S. Mesquita is grateful to CNPq and INPA by scientific initiation grants and CAPES for masters scholarship received. E. S. de Souza and M. I. C. Ozório thank FAPEAM by exchanges of technical support. C. V. Nunez thanks the PPBio/CNPq, FAPEAM and CT-Agro/CNPq for financial support.
REFERENCES
Grayer R.J., Kokubun T. 2001. Plant-fungal interactions: the search for phytoalexins and
other antifungal compounds from higher plants. Phytochemistry 56 (3): p 253-263.
Kraft C., Janett-Siems K., Siems K., Gupta M.P., Bienzle U., Eich E. 2000. Antiplasmodial
activity of isoflavones from Andira inermis. Journal of Ethnopharmacology 73 (1/2): p
131-135.
McLaughlin J.L., Saizarbitoria T.C., Anderson J.E. 1995. Tres bioensayos simples para
quimicos de productos naturales. Revista de la Sociedad Venezoelana de Quimica 18: p
13-18.
Mesquita, D. W. O., Mesquita, A. S. S., Cursino, L. M. C., Souza, E. S., Oliveira, A. C., Pinheiro, C. C. S., Novaes, J. A. P., Oliveira, J. A. A., Nunez, C. V. 2015. Atividades biológicas de espécies amazônicas de Rubiaceae. Revista Brasileira de Plantas Medicinais, 17.
Meyer B.N., Ferrigni N.R., Putnam J.E., Jacobsen L.B., Nichols D.E., McLaughlin J.L. 1982. Brine shrimp, a convenient general bioassy for active-plant constituents. Planta Medica45: p 31-34.
Parra A.L., Yhebra R.S., Sardinas I.G., Buelas L.I. 2001. Comparative study of the assay of
Artemia salina L. and the estimate of the medium lethal dose (LD50 value) in mice, to
determine oral acute toxicity of plant extracts. Phytomedicine 8 (5): p 395-400.
Peet, N.P., 2010. Drug resistance: a growing problem Drug Discovery Today 15 (15/16):
583-586.
Raven P.H., Evert R.F., Eichhorn S.E. 1996. Diversidade. In: Biologia Vegetal, Rio de Janeiro, Ed. Guanabara Koogan, p 157-407.
